LV-pIN-KDEL: a novel lentiviral vector demonstrates the morphology,dynamics and continuity of the endoplasmic reticulum in live neurones

Background  

The neuronal endoplasmic reticulum (ER) is an extensive, complex endomembrane system, containing Ca²⁺ pumps, and Ca²⁺ channels that permit it to act as a dynamic calcium store. Currently, there is controversy over the continuity of the ER in neurones, how this intersects with calcium signalling and the possibility of physical compartmentalisation. Unfortunately, available probes of ER structure such as vital dyes are limited by their membrane specificity. The introduction of ER-targeted GFP plasmids has been a considerable step forward, but these are difficult to express in neurones through conventional transfection approaches. To circumvent such problems we have engineered a novel ER-targeted GFP construct, termed pIN-KDEL, into a 3^rd generation replication-defective, self-inactivating lentiviral vector system capable of mediating gene transduction in diverse dividing and post-mitotic mammalian cells, including neurones.     

Analysis of glycoprotein processing in the endoplasmic reticulum using synthetic oligosaccharides

Protein quality control (QC) in the endoplasmic reticulum (ER) comprises many steps, including folding and transport of nascent proteins as well as degradation of misfolded proteins. Recent studies have revealed that high-mannose-type glycans play a pivotal role in the QC process. To gain knowledge about the molecular basis of this process with well-defined homogeneous compounds, we achieved a convergent synthesis of high-mannose-type glycans and their functionalized derivatives. We focused on analyses of UDP-Glc: glycoprotein glucosyltransferase (UGGT) and ER Glucosidase II, which play crucial roles in glycoprotein QC; however, their specificities remain unclear. In addition, we established an in vitro assay system mimicking the in vivo condition which is highly crowded because of the presence of various biomacromolecules.     

The evidence of Tobacco rattle virus impact on host plant organelles ultrastructure

Tobraviruses, like other (+) stranded RNA viruses of plants, replicate their genome in cytoplasm and use such usual membranous structures like endoplasmic reticulum. Based on the ultrastructural examination of Tobacco rattle virus (TRV)-infected potato and tobacco leaf tissues, in this work we provide evidence of the participation of not only the membranous and vesicular ER structures but also other cell organelles during the viral infection cycle. Non-capsidated TRV PSG particles (potato isolate from the Netherlands) (long and short forms) were observed inside the nucleus while the presence of TRV capsid protein (CP) was detected in the nucleus caryolymph and within the nucleolus area. Both capsidated and non-capsidated viral particles were localized inside the strongly disorganized chloroplasts and mitochondria. The electron-dense TRV particles were connected with vesicular structures of mitochondria as well as with chloroplasts in both potato and tobacco tissues. At 15–30 days after infection, vesicles filled with TRV short particles were visible in mitochondria revealing the expanded cristae structures. Immunodetection analysis revealed the TRV PSG CP epitope inside chloroplast with disorganized thylakoids structure as well as in mitochondria of different tobacco and potato tissues. The ultrastructural analysis demonstrated high dynamics of the main cell organelles during the TRV PSG–Solanaceous plants interactions. Moreover, our results suggest a relationship between organelle changes and different stages of virus infection cycle and/or particle formation.     

Electrical metal contacts to atomically thin 2H-phase MoTe2 grown by metal–organic chemical vapor deposition

We grow atomically thin molybdenum ditelluride (MoTe₂) films on a SiO₂/Si substrate by means of metal–organic chemical vapor deposition (MOCVD). Our Raman spectroscopy measurements reveal the formation of 2H-phase MoTe₂ films. Further, transmission electron microscopy and X-ray photoelectron spectroscopy studies indicate a three-atomic-layer structure and the surface element composition of MoTe₂ films. In this study, we mainly focus on the influence of metal contacts attached to the films on their electrical performance. We fabricate 2H-phase-MoTe₂-based field-effect transistors (FETs) with various metal contacts such as titanium/gold, nickel and palladium, which present p-type semiconductor properties. We also examine the influence of the work functions of the contact metals on the electrical properties of three-atomic-layer-MoTe₂-based FET devices. For a p-type MoTe₂ semiconductor, higher work functions of the contact metals afford narrower Schottky barrier heights (SBHs) and eventually highly efficient carrier injection through the contacts.     

Ultrastructural analysis and immunocytochemical localization of isolectins in Cratylia mollis seeds

Cratylia mollis is a native forage from the semi-arid region of Northeast, State of Pernambuco, Brazil, whose seeds have been considered an important lectin source. Multiple molecular forms of lectins, carbohydrate-binding proteins, have been purified from C. mollis seeds (Cra Iso) allowing several applications of these purified proteins. In this work seeds were processed for ultrastructural analysis and immunocytochemical localization of the two most abundant isolectins, Cra Iso 1 and Cra Iso 3, with glucose/mannose and galactose specificities, respectively. The ultrastructural analysis revealed a typical plant cell: organelles, nucleus and cellular wall were visualized. The localization of isolectins occurred mainly in the amorphous matrix of protein bodies, and in the cellular walls of the embryonic axis. The results showed that the isolectins, which differ in relation to carbohydrate specificity and glycosylation are located in the same cellular compartment suggesting different functions inside the same subcellular organelle. Cra Iso 1 and Cra Iso 3 distribution in the C. mollis seeds was consistent with the subcellular localization of several legume lectins.     

Current-dependent positive magnetoresistance in La_(0.8)Ba_(0.2)MnO_3 ultrathin films

We report an investigation into the magnetoresistance(MR) of La_(0.8) Ba_(0.2) Mn O_3 ultrathin films with various thicknesses. While the 13 nm-thick film shows the commonly reported negative magnetoresistive effect, the 6 nm-and 4 nmthick films display unconventional positive magnetoresistive(PMR) behavior under certain conditions. As well as the dependence on the film's thickness, it has been found that the electrical resistivity and the PMR effect of the thinner films are very dependent on the test current. For example, the magnetoresistive ratio of the 4 nm-thick film changes from +46%to-37% when the current is increased from 10 n A to 100 n A under 15 k Oe at 40 K. In addition, the two thinner films present opposite changes in electrical resistivity with respect to the test current, i.e., the electroresistive(ER) effect, at low temperatures. We discuss the complex magnetoresistive and ER behaviors by taking account of the weak contacts at grain boundaries between ferromagnetic metallic(FMM) grains. The PMR effect can be attributed to the breaking of the weak contacts due to the giant magnetostriction of the FMM grains under a magnetic field. Considering the competing effects of the conductive filament and local Joule self-heating at grain boundaries on the transport properties, the dissimilar ER effects in the two thinner films are also understandable. These experimental findings provide an additional approach for tuning the magnetoresistive effect in manganite films.     

基于接触数引导的迭代多组独立分子动力学模拟实现配体-蛋白质结合与解离过程

配体的结合与解离过程在蛋白质实现其生物学功能方面非常关键,因此对这些高度动态过程的研究变得非常重要. 尽管已有实验方法可以确定蛋白质-配体复合物的三维结构,但一般仅可获得静态图片. 随着计算机算力的快速提高以及算法的优化,分子动力学模拟在探索配体的结合与解离过程方面具有诸多优势. 然而,当系统变得足够大时,分子动力学模拟的时间和空间尺度成为了巨大的挑战. 本工作提出了一种研究配体-蛋白质结合与解离的增强采样工具,它基于配体和蛋白质之间形成的接触数来引导迭代多组独立分子动力学模拟. 在腺苷酸激酶的模拟结果中,观测到配体的结合和解离过程,而使用传统分子动力学模拟在同一时间尺度下则无法实现这一过程.     

The problems in quantum foundations in the light of gauge theories

We review the issues of nonseparability and seemingly acausal propagation of information in EPR, as displayed by experiments and the failure of Bell's inequalities. We show that global effects are in the very nature of the geometric structure of modern physical theories, occurring even at the classical level. The Aharonov-Bohm effect, magnetic monopoles, instantons, etc. result from the topology and homotopy features of the fiber bundle manifolds of gauge theories. The conservation of probabilities, a supposedly highly quantum effect, is also achieved through global geometry equations. The EPR observables all fit in such geometries, and space-time is a truncated representation and is not the correct arena for their understanding. Relativistic quantum field theory represents the global action of the measurement operators as the zero-momentum (and therefore spatially infinitely spread) limit of their wave functions (form factors). We also analyze the collapse of the state vector as a case of spontaneous symmetry breakdown in the apparatus-observed state interaction.Work supported in part by U.S. DOE Grant DE-FG05-85ER40200.Wolfson Chair Extraordinary in Theoretical Physics, TAUP N-161-85.     

Neuronal dysfunction with aging and its amelioration

The author focused on the functional decline of synapses in the brain with aging to understand the underlying mechanisms and to ameliorate the deficits. The first attempt was to unravel the neuronal functions of gangliosides so that gangliosides could be used for enhancing synaptic activity. The second attempt was to elicit the neuronal plasticity in aged animals through enriched environmental stimulation and nutritional intervention. Environmental stimuli were revealed neurochemically and morphologically to develop synapses leading to enhanced cognitive function. Dietary restriction as a nutritional intervention restored the altered metabolism of neuronal membranes with aging, providing a possible explanation for the longevity effect of dietary restriction. These results obtained with aging and dementia models of animals would benefit aged people.     

The neuronal ceroid lipofuscinosis <Emphasis Type="Italic">Cln8</Emphasis> gene expression is developmentally regulated in mouse brain and up-regulated in the hippocampal kindling model of epilepsy

Background  

The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative disorders characterized by accumulation of autofluorescent material in many tissues, especially in neurons. Mutations in the CLN8 gene, encoding an endoplasmic reticulum (ER) transmembrane protein of unknown function, underlie NCL phenotypes in humans and mice. The human phenotype is characterized by epilepsy, progressive psychomotor deterioration and visual loss, while motor neuron degeneration (mnd) mice with a Cln8 mutation show progressive motor neuron dysfunction and retinal degeneration.     

High-resolution nuclear track mapping in detailed cellular histology using CR-39 with the contact microscopy technique

In radiation cancer therapies using energetic charged particles such as proton/heavy-ion therapy and boron neutron capture therapy (BNCT), studies on radiation-induced biological response at cellular level are important because the radiation damage from energetic charged particles is highly localized along their paths and the radiation sensitivity is quite different in each cellular organelle. In such studies the information on the position of charged particle impact in biological cells is necessary. A novel method for high-resolution nuclear track mapping in detailed cellular histology has been developed. In this technique, biological specimens mounted on CR-39 plates are exposed to energetic charged particles. The irradiated samples are exposed to UV, and then etched for a short time. Both etch pits of nuclear tracks and relief for transmission UV image of the specimen can be observed on the CR-39 surface with an atomic force microscope (AFM) at about 100 nm resolution.     

Compartment-specific q-space analysis of diffusion-weighted data from isolated rhesus optic and sciatic nerves

We investigated compartment-specific water diffusion properties in two widely structurally different isolated bovine nerves. Sciatic and optic nerves were immersed in saline containing Gd-DTPA(2+). Consequently, T(1) became non-monoexponential and fit well to a biexponential function. q-Space diffusion data were collected for each component. In the sciatic nerve, the slow-decaying component (T(1s)) was considerably more restricted and directional than the fast-decaying component (T(1f)). In the optic nerve, fractional anisotropy of both components was comparable and similar to that of the total H(2)O signal. The root mean square of the displacement distribution functions of T(1s) correlated well with the widely different axonal diameters of both nerves. Possibly, the source of T(1s) is the intra-axonal compartment and that of T(1f) is associated with the inter-axonal space. The compartment specificity of the method shown makes it useful for the investigation of the contribution of each nerve compartment to diffusion tensor imaging measurements and other diffusion-based methods.     

激光细胞微手术的发展和应用

现代生命科学的发展在很大程度上依赖于对活细胞的加工和操纵,比如切除细胞内的某些特定蛋白或细胞器等.激光细胞微手术是一个强有力的工具,近年来在细胞微手术精度控制方面取得很大的进展.综述了激光细胞微手术的最新发展及其在生物学和生物医学工程中的应用,对目前激光细胞微手术中存在的主要问题进行了讨论,对比了普通激光聚焦法、飞秒激...     

Production cross sections for the heaviest nuclei in complete fusion reactions induced by heavy ions

Production of the heaviest nuclei in complete fusion reactions induced by heavy ions has been considered in a systematic way in the framework of the conventional barrier passing model coupled with the statistical model. Available data on excitation functions for fission and production of evaporation residues (ER) in very asymmetric combinations induced by ions lighter than Ne on actinide target nuclei are described rather well in the framework of these models. The data allow one to adjust model parameters and to reveal the quasi-fission effect caused by the interaction with deformed target nuclei, which is manifested in the suppression of the ER production at sub-barrier energies. For reactions induced by Mg and heavier projectiles, quasi-fission is starting to suppress fusion (ER production) at energies above the Coulomb barrier. One has to introduce empirically the quantity of the fusion probability P_fus to reproduce the ER excitation functions in the framework of the conventional approach. The exponential dependence of P_fus on the combined fissility parameter (a similar parameter that was introduced for the extra-push energy scaling) was found in search for scaling for the P_fus values resulting from the data analysis.     

Deconvolution of compartmental water diffusion coefficients in yeast-cell suspensions using combined T(1) and diffusion measurements

An NMR method is presented for measuring compartment-specific water diffusion coefficient (D) values. It uses relaxography, employing an extracellular contrast reagent (CR) to distinguish intracellular (IC) and extracellular (EC) (1)H(2)O signals by differences in their respective longitudinal (T(1)) relaxation times. A diffusion-weighted inversion-recovery spin-echo (DW-IRSE) pulse sequence was used to acquire IR data sets with systematically and independently varying inversion time (TI) and diffusion-attenuation gradient amplitude (g) values. Implementation of the DW-IRSE technique was demonstrated and validated using yeast cells suspended in 3 mM Gd-DTPA(2-) with a wet/dry mass ratio of 3.25:1.0. Two-dimensional (2D) NMR data were acquired at 2.0 T and analyzed using numerical inverse Laplace transformation (2D- and sequential 1D-ILT) and sequential exponential fitting to yield T(1) and water D values. All three methods gave substantial agreement. Exponential fitting, deemed the most accurate and time efficient, yielded T(1):D (relative contribution) values of 304 ms:0.023x10(-5) cm(2)/s (47%) and 65 ms:1.24x10(-5) cm(2)/s (53%) for the IC and EC components, respectively. The compartment-specific D values derived from direct biexponential fitting of diffusion-attenuation data were also in good agreement. Extension of the DW-IRSE method to in vivo models should provide valuable insights into compartment-specific water D changes in response to injury or disease. (c) 2002 Elsevier Science (USA).     

Shot noise by quantum scattering in chaotic cavities

We have experimentally studied shot noise of chaotic cavities defined by two quantum point contacts in series. The cavity noise is determined as (1/4)2e/I/ in agreement with theory and can be well distinguished from other contributions to noise generated at the contacts. Subsequently, we have found that cavity noise decreases if one of the contacts is further opened and reaches nearly zero for a highly asymmetric cavity. Heating inside the cavity due to electron-electron interaction can slightly enhance the noise of large cavities and is also discussed quantitatively.     

Ohmic Contacts to ZnSe-Based Materials

The formation of ohmic contacts to n- and p-type ZnSe is reviewed. The mechanisms for forming reasonable low-resistance ohmic contacts to n-ZnSe are well understood. This results from the fact that the Fermi energy level of ZnSe is unpinned and metals with sufficiently large work functions can make contact to n-type material. However, the situation is reversed for p-ZnSe where a large band gap and large electron affinity make it impossible to find metals with sufficiently large work functions to create an ohmic contact. Instead, the use of HgSe to form low barrier height Schottky contacts and of ZnSe/ZnTe multiple quantum wells (MQWs) to form ohmic contacts is reviewed. Although the MQWs can be used to form ohmic contacts to p-ZnSe, they degrade at high temperatures and high current densities. This is reviewed and shown to be a serious problem for applications to laser diodes.